Paclitaxel (TAXOL®) is an important anti-cancer drug initially isolated from Taxus brevifolia. It is a complex diterpenoid alkaloid found in several Taxus species (Wani et al., J. Am. Chem. Soc. 93: 2325-2327, 1971; Vidensek et al., J. Nat. Prod. 53: 1609-1610, 1990; Witherup et al., J. Nat. Prod. 53: 1249-1255, 1990; Mattina & Paiva, J. Environ. Hortic. 10: 187-191, 1992; Wheeler et al., J. Nat. Prod. 55: 432-440, 1992; Choi et al., Planta Med. 61: 264-266, 1994; Wickremesinhe & Arteca, Plant Sci. 101: 15-135, 1994; and Kwak et al., Phytochemistry 40: 29-32, 1995). Paclitaxel has the unique mechanism of action for stabilizing microtubules against depolymerization (Seki et al., J. Chem. Eng. Jpn. 28: 488-490, 1995). Microtubules are tubular protein polymers composed of two tubulin polypeptides; their dynamic behavior is important in cell proliferation. Paclitaxel blocks mitosis at the transition between the metaphase and anaphase by stabilizing the microtubules, which subsequently induces cell death (Jordan & Wilson, In Taxane anticancer agents (eds.) Georg et al., ACS symposium Series 583. American Chemical Society, Washington, D.C. pp. 138-153, 1995). The clinical trials of paclitaxel in patients with various types of cancers showed antineoplastic activities against ovarian, breast, lung, head, neck and gastrointestinal cancers (Holmes et al., In Taxane anticancer agents. Georg et al. (eds.) ACS symposium Series 583. American Chemical Society. Washington D.C. pp. 31-57, 1995).
The broad spectrum anticancer activity of paclitaxel accounts for its great demand in the pharmaceutical industry world wide. Taxus trees are grown in commercial nurseries, and the needles and stems are used to extract taxanes which are used as semi-synthetic sources of paclitaxel (Joyce, Bioscience 43: 133-136, 1993; Wheeler & Hehnen, J. For. 91: 15-18, 1993). However, the supply of the drug is limited as the Taxus species are slow growing gymnosperms and the content of paclitaxel in the bark of the trees is relatively low (0.01% on dry weight basis). The conventional breeding strategy for genetic improvement of Taxus is not a feasible approach due to the heterozygosity of the genome, slow growth and long generation time of the species. Cell and tissue culture techniques offer an alternative system for paclitaxel production. In the 1950s, La Rue (In Abnormal and pathological plant growth, Report Symp. Brookhaven National Laboratory, J Upton, N.Y., pp 187-208, 1953) and Tulecke (Tulecke, Bull. Torry Bot. Club. 86: 283-289, 1959) initiated approaches to Taxus plant cell and tissue culture. The Taxus cells are known to grow slowly during initial subcultures, tend to turn brown within 8-10 months and growth stops. But recovery of fresh cells from brown callus has been reported after 1-2 years (Gibson et al., In Taxol: Science and Application, Stuffness (ed.) pp. 71-95. Boca Raton, N.Y.: CRC Press, 1995). At present, the production capability of paclitaxel and related taxanes using cell and tissue culture has been established and the conditions suitable for fast growing cultures to produce high levels of paclitaxel have been studied in commercial production (Takeya, In Taxus, Itokawa and Lee (eds). Taylor and Francis Group, London and New York. Pp 134-150, 2003). However the low productivity of paclitaxel combined with consistent variability is a major concern in realizing the full potential of producing this drug in cell cultures. Metabolic engineering appears an attractive route to enhance the paclitaxel production in cell cultures especially with the elucidation of the paclitaxel biosynthetic pathways in the recent past (Ketchum & Croteau, In Towards Natural Medicine Research in the 21st Century. Ageta et al. (eds.), Elsevier Sciences B. V. pp 339-348, 1998). The success of this approach is dependent on the establishment of a viable transformation system in Taxus. Although several conifers have been transformed by Agrobacterium (Sederoff et al., Bio/Technology 4: 647-649, 1986; Loopstra et al., Plant Mol. Biol. 15:1-9, 1990; and Huang et al., In Vitro Cell. Dev. Biol. 27: 201-207, 1991) and particle bombardment (Robertson et al., Plant Mol. Biol. 19: 925-935, 1992; Ellis et al., Bio/Technology 11: 84-89, 1993), there is very limited success in Taxus transformation. Tumor induction using wild strains of agrobacterium tumefaciens has been reported in Taxus brevifolia and Taxus baccata (Han et al., Plant Sci. 95: 187-196, 1994; Han et al., In Biotechnology in Agriculture and Forestry, Vol. 44 Transgenic Trees. Bajaj (ed.) Chapter XXI. pp 291-306, 1999). Transient expression of Gus reporter gene in zygotic embryos of Taxus brevifolia was reported by Luan et al. (In Vitro Cell. Dev Biol Plant 32: 81-85, 1996). Similar transient expression of GFP (green fluorescent protein) was obtained in Taxus cuspidata callus, that was sustained for three months in culture (Kim et al., J. Microbiol. Biotechnol. 10: 91-94, 2000). The use of Agrobacterium rhizogenes for hairy root induction was yet another attempt towards transformation of the Taxus species (Huang et al., Yunnan Zhiwu Yanjiu, 19: 292-296, 1997; Zunxi et al., Acta Botanica Yunnanica 19: 292-296, 1997).
Development of a stable transformation system for Taxus is critical for enhanced production of paclitaxel by genetic engineering strategy.